"Frontmatter". In: Plant Genomics and Proteomics

Partial cDNA
CAP
B
B
AAAAAAAA
XTTTTTTTTTT
RE site primer adapter
Protected CAP
RNase sensitive CAP - removed
AAAAAAAA
XTTTTTTTTT
RE site primer adapter
AAAAAAAAA
XTTTTTTTTTT
RE site primer adapter
XTTTTTTTTTTT
RE site primer adapter
XTTTTTTTTTT
RE site primer adapter
GGGGGGGGGG
GGGGGGGGGG
Second primer adapter 
CCCCCCCCCC
AAAAAAAA
XTTTTTTTTTT
RE site primer adapter
CAP
B
B
CAP
B
S
Full length cDNA clones
FIGURE 4.3.
Schematic representation of a general strategy to select and clone full-
length cDNAs: Biotinylation (B) of diol groups and synthesis of first-strand cDNA
with a degenerate primer adapter including restriction enzyme (RE) site; RNase I
digestion; full-length cDNA captured by streptavidin-coated magnetic beads (S);
cDNA released from beads by RNA hydrolysis; first-strand cDNA tailed with
oligo(dG); second-strand cDNA synthesis primed; cDNA cloned using the restriction
sites introduced with the primers (Adapted from LabLink 2, No. 1 1998
http://www.cpg-biotech.com/lablink/lablink_2_1_1.html).

---

annotation and for the identification of alternative splicing sites for RNAs.
Three possible strategies for full-length sequencing are:
∑ Transposon mutagenesis: A transposon is randomly inserted, in vitro,
into the cDNA insert, and primers designed from both sides of the
transposon are used for sequencing. Sequencing a number of inde-
pendent transposon sites will be sufficient to assemble the complete
cDNA sequence.
∑ Concatenated cDNA sequencing (CCS): cDNA inserts are concate-
nated into a BAC-size molecule (100 kb) and then sheared, cloned,
and shotgun sequenced by standard procedures. The full-length
cDNA sequences are then assembled and edited with the appropri-
ate software.
∑ Primer walking: Primers are designed from 5¢ and 3¢ end sequences
and used for a second round of sequencing. Additional primers are
then made and used until the whole contiguous cDNA sequence is
obtained.
The availability of full-length cDNA clones opens new avenues for iden-
tifying gene function and protein interactions. As detailed more fully in
Chapter 6, the use of tagged full-length cDNAs in transgenic plants can be
a first step in isolating and identifying the protein complexes that exist in
vivo. Transgenics can also be used to develop a protein atlas of where in the
cell each of the genes is expressed. A full-length cDNA can be tagged with
a dye and the tagged probe transformed back into the plant under the control
of its native promoter. The site of the fluorescence will indicate the organ or
tissue where the gene is expressed, as well as the cellular localization of the
protein. The reintroduction of the full-length cDNA into a plant can also
result in either overexpression or silencing of that gene. The subsequent 
phenotype that is observed provides clues as to the function of the gene. In
addition, overexpression of such a gene, for which a full-length cDNA is
available, can be accomplished in a heterologous system, such as yeast or 
E. coli, followed by in vitro studies of the protein function. 
Thus, although ESTs are useful for the initial description of the tran-
scriptome, the full-length cDNAs are a much more valuable resource. The
availability of full-length cDNAs for many species will greatly enhance the
rate of gene discovery, patterns of splicing, and the understanding of gene
function in plants.